The widely abused amphetamine analogue, 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) selectively damages the axon terminals of 5-HT neurons in the brain. Several lines of evidence suggest that dopamine (DA) contributes to this toxicity. Although DA clearly produces oxidative by-products such as reactive oxygen species and quinones, it remains unclear as to how DA-derived oxidative species produce selective damage to 5HT terminals, a hallmark of MDMA-induced neurotoxicity. More specifically, the mechanism as to how DA accumulates within 5-HT terminals to produce its selective damage is unknown. The overaraching hypothesis of the current proposal is that L- tyrosine, the amino acid precursor of DA, contributes to the neurodegenerative process. This hypothesis is based on our recent preliminary data indicating a 5-fold increase in the extracellular concentration of tyrosine measured in vivo after MDMA. Unlike DA, tyrosine is actively transported from the periphery and into the brain and neurons. While tyrosine is the natural precursor for DA synthesis within DA neurons, high concentrations of tyrosine in 5HT neurons may have deleterious consequences. The hypothetical framework of this proposal is that the oxidative environment produced by MDMA and hyperthermia in 5-HT neurons causes the non-enzymatic oxidation of tyrosine to the DA precursor, DOPA. Aromatic amino acid decarboxylase (AADC), within the 5-HT terminal then would decarboxylate DOPA to DA, leading to an accumulation of DA and consequently, DA-derived reactive oxygen species and oxidative damage within 5HT terminals. This hypothesized role of tyrosine as a mediator of MDMA-induced toxicity is a novel mechanism that effectively synthesizes current existing hypotheses and to some extent, discrepant observations and apparent caveats into a cohesive, theoretical and testable framework. The Specific Aims are (1) to demonstrate the non-enzymatic oxidation of tyrosine to DOPA and DA within 5HT neurons, (2) to characterize the contributions of L-tyrosine and tyramine to MDMA and hyperthermia-induced oxidative stress and (3) to assess subsequent neuronal damage. The use of cultured RN46A 5HT cells is a novel approach that is uniquely suited to address the hypothesized mechanism of MDMA-induced damage. These experiments will directly measure the individual and relative effects of MDMA, tyrosine, hyperthermia, and the formation of intracellular dopamine on oxidative processes within 5-HT neurons and how these variables affect cell viability. The testing of this model in conjunction with in vivo microdialysis studies, provides a unique and powerful approach to address enigmatic issues previously related to MDMA-induced neurodegeneration of 5HT systems.